scholarly journals Isolation of a lipid a bound polypeptide responsible for LPS- initiated mitogenesis of C3H/HeJ spleen cells

1976 ◽  
Vol 144 (3) ◽  
pp. 840-846 ◽  
Author(s):  
DC Morrison ◽  
SJ Betz ◽  
DM Jacobs
Keyword(s):  
Lipid A ◽  
Critical Role ◽  
Gel Filtration ◽  
Polymyxin B ◽  
Mitogenic Activity ◽  
Partial Purification ◽  
Spleen Cells ◽  
Phenol Treatment ◽  
Bacterial Lipopolysaccharides

The experiments by Sultzer and Nilsson (1), and later by Watson and Riblet (2), established that spleen cells from the C3H/HeJ strain of mouse were refractory to the mitogenic effects of bacterial lipopolysaccharides (LPS). More recently, however, experiments from our laboratory (3) demonstrated that spleen cells from C3H/HeJ mice were in fact responsive to some preparations of LPS but not to others, and that the method of extraction played a critical role in determining activity. In particular, preparations of LPS prepared by extraction with aqueous butanol had potent mitogenic activity. Our data showed that the mitogenic activity of such positive preparations of LPS coisolated with the LPS during gel filtration chromatography and subsequent equilibrium banding on CsCl. In addition, lipid A isolated from positive preparations of LPS was also capable of stimulating C3H/HeJ spleen cells. Taken together, these experiments provided rather convincing data that it was the LPS (in particular the lipid A) itself, or some contaminant very tightly bound to the lipid A, which was responsible for its biological activity. We further demonstrated that treatment of positive preparations of LPS with hot phenol rendered such preparations nonmitogenic for C3H/HeJ spleens, yet activity for other strains was only moderately decreased. These experiments would suggest either that the phenol treatment chemically alters the lipid A region of the LPS molecule or that such treatment removes the putative tightly bound contaminant responsible for C3H/HeJ mitogenesis. In the experiments reported here, we have explored in greater detail the role of lipid A in the stimulation of C3H/HeJ spleen cells. For these experiments we have utilized our earlier observations that the antibiotic polymyxin B forms a highly stable molecular complex with the lipid A region of LPS (4), and that such polymyxin B-LPS complexes are unable to mitogenically stimulate B lymphocytes (5). In addition, we have attempted to distinguish between the two potential modes of action of phenol on LPS, namely, the chemical alteration of the lipid A or the removal of a tightly bound contaminant by phenol treatment. The results of the experiments we report here support the interpretation that mitogenic activity of positive preparations of LPS is associated with a low mol wt phenol soluble polypeptide of approximately 10,000 mol wt. After partial purification, this polypeptide intitiates a significant mitogenic response at concentrations as low as 10 μg/ml. We conclude that the C3H/HeJ strain of mouse is a true nonresponder to the stimulatory effects of the lipid A region of LPS.

The mitogenic activity of lipopolysaccharide for spleen cells from germfree, conventional, and gnotobiotic rats

1979 ◽  
Vol 25 (9) ◽  
pp. 1087-1093 ◽  
Author(s):  
Carol Wells ◽  
Edward Balish
Keyword(s):  
Spleen Cell ◽  
Lipid A ◽  
Mitogenic Activity ◽  
Adherent Cells ◽  
Spleen Cells ◽  
E Coli ◽  
Blastogenic Response ◽  
Pseudomonas Species ◽  
Nonadherent Cells ◽  
Germfree Rats

Spleen cells from germfree rats, conventionally reared rats, and gnotobiotic rats associated with two Pseudomonas species gave no positive blastogenic response when incubated with each of four lipopolysaccharide (LPS) preparations from Escherichia coli, with glycolipid extracted from Salmonella minnesota R595 or with S. minnesota R595 lipid A. However, spleen cell preparations from athymic mice demonstrated a positive blastogenic response when incubated with E. coli LPS. Removal of adherent cells from germfree and conventional-flora rat spleen cells did not increase the mitogenic activity of LPS for nonadherent cells (< 0.5% esterase-positive cells). All rat spleen cell preparations gave positive blastogenic responses to phytohemagglutinin and concanavalin A. This study indicates that LPS may not be a mitogenic agent for rat spleen cells.

Clearance of bacterial lipopolysaccharides and lipid A by the liver and the role of arginino-succinate synthase

2008 ◽  
Vol 14 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Motonobu Satoh ◽  
Shingo Ando ◽  
Takehiro Shinoda ◽  
Masatoshi Yamazaki
Keyword(s):  
Lipid A ◽  
Bacterial Lipopolysaccharides

scholarly journals Partial purification and characterization of a peroxidase activity from human placenta

1990 ◽  
Vol 268 (3) ◽  
pp. 739-743 ◽  
Author(s):  
J L Nelson ◽  
A P Kulkarni
Keyword(s):  
Peroxidase Activity ◽  
Potential Role ◽  
Gel Filtration ◽  
Partial Purification ◽  
Human Haemoglobin ◽  
Prostaglandin Synthase ◽  
Membrane Bound ◽  
Cytochrome P 450

Peroxidases can metabolize a variety of xenobiotics to reactive intermediates capable of binding to protein or DNA. The potential role of these enzymes in fetotoxicity has not been explored. In this study, the presence of peroxidase activity was observed in human term and pre-term placenta. Human term placental peroxidase activity (HTPP) was partially purified by concanavalin A affinity chromatography from CaCl2 extracts of the particulate fraction. HTPP appears to be a membrane-bound glycoprotein. Arachidonic acid-dependent oxidation of guaiacol was not observed, suggesting that the peroxidase activity was not due to prostaglandin synthase. Moreover, HTPP preparations were devoid of catalase and spectrally dissimilar from human haemoglobin, cytochrome P-450, eosinophil peroxidase and myloperoxidase, suggesting an endogenous origin. An Mr of approx. 119,000 was determined for HTPP by gel filtration. Cathodic slab-PAGE of cetyltrialkylammonium bromide-solubilized HTPP yielded two peroxidase-staining bands.

Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides

1976 ◽  
Vol 13 (10) ◽  
pp. 813-818 ◽  
Author(s):  
David C. Morrison ◽  
Diane M. Jacobs
Keyword(s):  
Lipid A ◽  
Polymyxin B ◽  
Bacterial Lipopolysaccharides

scholarly journals Role of Lipid A Acylation in Yersinia enterocolitica Virulence

2010 ◽  
Vol 78 (6) ◽  
pp. 2768-2781 ◽  
Author(s):  
Camino Pérez-Gutiérrez ◽  
Enrique Llobet ◽  
Catalina M. Llompart ◽  
Mar Reinés ◽  
José A. Bengoechea
Keyword(s):  
Virulence Factors ◽  
Yersinia Enterocolitica ◽  
Lipid A ◽  
Polymyxin B ◽  
Human Pathogen ◽  
Present Evidence ◽  
Host Environment ◽  
Serotype O

ABSTRACT Yersinia enterocolitica is an important human pathogen. Y. enterocolitica must adapt to the host environment, and temperature is an important cue regulating the expression of most Yersinia virulence factors. Here, we report that Y. enterocolitica 8081 serotype O:8 synthesized tetra-acylated lipid A at 37°C but that hexa-acylated lipid A predominated at 21°C. By mass spectrometry and genetic methods, we have shown that the Y. enterocolitica msbB, htrB, and lpxP homologues encode the acyltransferases responsible for the addition of C12, C14 and C16:1, respectively, to lipid A. The expression levels of the acyltransferases were temperature regulated. Levels of expression of msbB and lpxP were higher at 21°C than at 37°C, whereas the level of expression of htrB was higher at 37°C. At 21°C, an lpxP mutant was the strain most susceptible to polymyxin B, whereas at 37°C, an htrB mutant was the most susceptible. We present evidence that the lipid A acylation status affects the expression of Yersinia virulence factors. Thus, expression of flhDC, the flagellar master regulatory operon, was downregulated in msbB and lpxP mutants, with a concomitant decrease in motility. Expression of the phospholipase yplA was also downregulated in both mutants. inv expression was downregulated in msbB and htrB mutants, and consistent with this finding, invasion of HeLa cells was diminished. However, the expression of rovA, the positive regulator of inv, was not affected in the mutants. The levels of pYV-encoded virulence factors Yops and YadA in the acyltransferase mutants were not affected. Finally, we show that only the htrB mutant was attenuated in vivo.

scholarly journals Characterization and Biological Role of the O-Polysaccharide Gene Cluster of Yersinia enterocolitica Serotype O:9

2007 ◽  
Vol 189 (20) ◽  
pp. 7244-7253 ◽  
Author(s):  
Mikael Skurnik ◽  
Marta Biedzka-Sarek ◽  
Peter S. Lübeck ◽  
Tea Blom ◽  
José Antonio Bengoechea ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Yersinia Enterocolitica ◽  
Lipid A ◽  
Inner Core ◽  
Attachment Site ◽  
Polymyxin B ◽  
Outer Core ◽  
Core Oligosaccharide ◽  
Serotype O

ABSTRACTYersinia enterocoliticaserotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) inY. enterocoliticaO:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer ofN-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of thegndgene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster,galFandgalU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.

A Critical Role for N- and O-Linked Carbohydrates In Modulating Von Willebrand Factor Clearance In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 382-382 ◽  
Author(s):  
Emily McRae ◽  
Orla Rawley ◽  
Hendrik Nel ◽  
Rachel Therese McGrath ◽  
Gudmundur Bergsson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Critical Role ◽  
Gel Filtration ◽  
Macrophage Depletion ◽  
Plasma Half Life

Abstract Abstract 382FN2 VWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF−/− mice. Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p<0.01). Digestion of pdVWF with α2–3 neuraminidase to remove predominantly O-linked sialic acid (which constitutes less than 20% total VWF sialylation) was also sufficient to markedly enhance VWF clearance (t1/2 = 13.1 min; p<0.05). In the presence of the asialoglycoprotein receptor (ASGPR)-antagonist ASOR, the mean residence time of Neu-VWF was identical to that of pd-VWF. Recent studies have shown that macrophages may be important in VWF clearance. Since the ASGPR is expressed on both hepatocytes and macrophages, the effect of macrophage depletion on VWF clearance was assessed. Pre-treatment with liposome-encapsulated clodronate depleted F4/80+CD11b+ murine macrophages by 75%, and significantly prolonged Neu-VWF survival. However Neu-VWF survival was not corrected to that observed in the presence of ASOR. For example, plasma Neu-VWF survival after 5 mins was corrected from 30±6% to 92±7% in the presence of ASOR, compared to 78±10% following clodronate macrophage-depletion. Cumulatively, these findings demonstrate that both N- and O-linked sialylation are critical in protecting VWF against ASGPR-mediated clearance. Moreover, ASGPR-modulated clearance is at least in part macrophage-dependent. ß-galactose residues exposed following removal of capping sialic acid are recognised by the ASGPR. To further define the role of specific sugars in regulating VWF clearance, the effect of terminal sialic acid and sub-terminal galactose removal by sequential neuraminidase and galactosidase digestions was studied. Surprisingly, VWF exposed to sequential neuraminidase and galactosidase digestions (NeuGal-VWF) was cleared rapidly from the plasma in a monophasic fashion (t1/2 = 4.8 min). Moreover, treatment with PNGase F to completely remove N-linked carbohydrate structures also markedly decreased the plasma half-life (PNG-VWF; t1/2 = 2.1 min). In keeping with their lack of exposed galactose residues, the enhanced clearance of NeuGal-VWF and PNG-VWF were not mediated via the ASGPR (ASOR had no significant effect). In contrast, macrophage depletion by liposomal clodronate significantly inhibited the enhanced clearance of both NeuGal-VWF and PNG-VWF respectively. These data suggest that the ASGPR is not the only macrophage receptor involved in modulating VWF clearance, which is consistent with the relatively minor prolongation in VWF survival previously reported in Asgpr1−/− mice. These novel data demonstrate that variation in the N- or O-linked carbohydrate structures significantly modulate VWF half-life in vivo. Moreover, VWF clearance is not mediated solely through the ASGPR, but may also require additional as yet unidentified macrophage receptors for full clearance. Therefore, qualitative and quantitative variation in VWF glycosylation represents a key regulator of VWF clearance, and as such is likely to be of direct pathophysiological significance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bioactive lipid: A novel diagnostic approach for retinoblastoma in clinical management

2021 ◽  
Vol 0 ◽  
pp. 1-4
Author(s):  
Ankit Srivastava ◽  
Bimal Prasad Jit ◽  
Rutumbara Dash ◽  
Manasa Kumar Panda
Keyword(s):  
Lysophosphatidic Acid ◽  
Lipid A ◽  
Critical Role ◽  
Bioactive Lipids ◽  
Cellular Functions ◽  
Epidemiological Evidence ◽  
Diagnostic Biomarkers ◽  
Sphingosine 1 Phosphate ◽  
Mechanistic Basis

Bioactive lipids, presumably lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), play a critical role in regulating an array of cellular functions ranging from cellular fate determination, inflammation, immunity, and cancer. Epidemiological evidence suggests that both the metabolites play a prominent role in the development and progression of oncogenic phenotype in a variety of cancers including breast, colorectal, pancreatic, and lymphoma. Previous studies have demonstrated the possible association of LPA, S1P and their receptor in regulating the pathogenesis of retinoblastoma, however, the exact mechanism involved in this event has not been studied in detail. Importantly, understating the mechanistic basis of LPA and S1P regulation is of utmost significance, as far the phenotypical complexity of retinoblastoma (RB) is concerned. Findings from the recent investigations elucidate the prospective role of S1P in provoking the chemoresistant behavior of RB cells for etoposide. In this context, the current paper will enable the identification of novel diagnostic biomarkers and therapeutic targets for better treatment and clinical efficacy in children with RB.

Lipid A dependence of the ocular response to circulating endotoxin in rabbits

1980 ◽  
Vol 30 (3) ◽  
pp. 786-790
Author(s):  
E L Howes ◽  
D C Morrison
Keyword(s):  
Vascular Permeability ◽  
Lipid A ◽  
Polymyxin B ◽  
High Lipid ◽  
Rough Strain ◽  
Direct Binding ◽  
Heart Blood ◽  
Bacterial Lipopolysaccharides ◽  
The One

The effects of bacterial lipopolysaccharides on ocular vascular permeability were measured after their intravenous injection in rabbits. Alterations in ocular vascular permeability were quantitated by the accumulation of 125I-labeled albumin in the enucleated eye compared with that in heart blood (ocular albumin space). Two lipopolysaccharides extracted from Escherichia coli O111:B4, one with high lipid A content and one with high polysaccharide content, were tested initially, and the one with greater lipid A was 200 times more effective in producing an alteration in ocular vascular permeability. Lipopolysaccharide from a rough strain, Salmonella minnesota (R595), containing lipid A primarily, as well as a purified lipid A extracted from +595, were also effective. But an extract of the protein associated with lipid A was without significant effect. In vitro pretreatment of the lipopolysaccharides with polymyxin B, an inhibitor of the biological activity of lipid A through direct binding, could abrogate the ocular response. These results indicate the paramount importance of the lipid A moiety in the ocular response to circulating endotoxin.

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